Growth factor

ABSTRACT

VEGF-D, a new member of the PDGF family of growth factors, which among other things stimulates endothelial cell proliferation and angiogenesis and increases vascular permeability, as well as nucleotide sequences encoding it, methods for producing it, antibodies and other antagonists to it, transfected or transformed host cells for expressing it, pharmaceutical compositions containing it, and uses thereof in medical and diagnostic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 09/296,275, filedApr. 22, 1999, which is a division of application Ser. No. 08/915,795,filed Aug. 21, 1997, now U.S. Pat. No. 6,235,713. This application alsoclaims the benefit of the filing dates of the following copending U.S.Provisional Application Ser. No. 60/023,751, filed Aug. 23, 1996; Ser.No. 60/031,097, filed Nov. 14, 1996; Ser. No. 60/038,814, filed Feb. 10,1997; and Ser. No. 60/051,426, filed Jul. 1, 1997.

FIELD OF THE INVENTION

This invention relates to growth factors for endothelial cells, and inparticular to a novel vascular endothelial growth factor, DNA encodingthe factor, and to pharmaceutical and diagnostic compositions andmethods utilizing or derived from the factor.

BACKGROUND OF THE INVENTION

Angiogenesis is a fundamental process required for normal growth anddevelopment of tissues, and involves the proliferation of newcapillaries from pre-existing blood vessels. Angiogenesis is not onlyinvolved in embryonic development and normal tissue growth, repair, andregeneration, but is also involved in the female reproductive cycle,establishment and maintenance of pregnancy, and in repair of wounds andfractures. In addition to angiogenesis which takes place in the normalindividual, angiogenic events are involved in a number of pathologicalprocesses, notably tumor growth and metastasis, and other conditions inwhich blood vessel proliferation, especially of the microvascularsystem, is increased, such as diabetic retinopathy, psoriasis andarthropathies. Inhibition of angiogenesis is useful in preventing oralleviating these pathological processes.

On the other hand, promotion of angiogenesis is desirable in situationswhere vascularization is to be established or extended, for exampleafter tissue or organ transplantation, or to stimulate establishment ofcollateral circulation in tissue infarction or arterial stenosis, suchas in coronary heart disease and thromboangitis obliterans.

Because of the crucial role of angiogenesis in so many physiological andpathological processes, factors involved in the control of angiogenesishave been intensively investigated. A number of growth factors have beenshown to be involved in the regulation of angiogenesis; these includefibroblast growth factors (FGFs), platelet-derived growth factor (PDGF),transforming growth factor α (TGFα), and hepatocyte growth factor (HGF).See, for example, Folkman et al., “Angiogenesis”, J. Biol. Chem., 1992267 10931-10934 for a review.

It has been suggested that a particular family of endothelialcell-specific growth factors and their corresponding receptors isprimarily responsible for stimulation of endothelial cell growth anddifferentiation, and for certain functions of the differentiated cells.These factors are members of the PDGF family, and appear to act viaendothelial receptor tyrosine kinases (RTKs). Hitherto four vascularendothelial growth factor subtypes have been identified. Vascularendothelial growth factor (VEGF), now known as VEGF-A, has been isolatedfrom several sources. VEGF-A shows highly specific mitogenic activityagainst endothelial cells, and can stimulate the whole sequence ofevents leading to angiogenesis. In addition, it has strongchemoattractant activity towards monocytes, can induce plasminogenactivator and plasminogen activator inhibitor in endothelial cells, andcan also influence microvascular permeability. Because of the latteractivity, it is also sometimes referred to as vascular permeabilityfactor (VPF). The isolation and properties of VEGF have been reviewed;see Ferrara et al., “The Vascular Endothelial Growth Factor Family ofPolypeptides”, J. Cellular Biochem., 1991 47 211-218 and Connolly,“Vascular Permeability Factor: A Unique Regulator of Blood VesselFunction”, J. Cellular Biochem., 1991 47 219-223.

More recently, three further members of the VEGF family have beenidentified. These are designated VEGF-B, described in InternationalPatent Application No. PCT/US96/02957 (WO 96/26736) by Ludwig Institutefor Cancer Research and The University of Helsinki, VEGF-C, described inJoukov et al., The EMBO Journal, 1996 15 290-298, and VEGF2, describedin International Patent Application No. PCT/US94/05291 (WO 95/24473) byHuman Genome Sciences, Inc. VEGF-B has closely similar angiogenic andother properties to those of VEGF, but is distributed and expressed intissues differently from VEGF. In particular, VEGF-B is very stronglyexpressed in heart, and only weakly in lung, whereas the reverse is thecase for VEGF. This suggests that VEGF and VEGF-B, despite the fact thatthey are co-expressed in many tissues, may have functional differences.

VEGF-B was isolated using a yeast co-hybrid interaction trap screeningtechnique, screening for cellular proteins which might interact withcellular retinoic acid-binding protein type I (CRABP-I). Its isolationand characteristics are described in detail in PCT/US96/02597 and inOlofsson et al., Proc. Natl. Acad. Sci., 1996 93 2576-2581.

VEGF-C was isolated from conditioned media of PC-3 prostateadenocarcinoma cell line (CRL1435) by screening for ability of themedium to produce tyrosine phosphorylation of the endothelialcell-specific receptor tyrosine kinase Flt-4, using cells transfected toexpress Flt-4. VEGF-C was purified using affinity chromatography withrecombinant Flt-4, and was cloned from a PC-3 cDNA library. Itsisolation and characteristics are described in detail in Joukov et al.,The EMBO Journal, 1996 15 290-298.

VEGF2 was isolated from a highly tumorgenic, estrogen-independent humanbreast cancer cell line. While this molecule is stated to have about 22%homology to PDGF and 30% homology to VEGF, the method of isolation ofthe gene encoding VEGF2 was unclear, and no characterization of thebiological activity was disclosed.

Vascular endothelial growth factors appear to act by binding to receptortyrosine kinases of the PDGF-receptor family. Five endothelialcell-specific receptor tyrosine kinases have been identified, namelyFlt-1 (VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt-4 (VEGFR-3), Tie andTek/Tie-2. All of these have the intrinsic tyrosine kinase activitywhich is necessary for signal transduction. The essential, specific rolein vasculogenesis and angiogenesis of Flt-1, Flk-1, Tie and Tek/Tie-2has been demonstrated by targeted mutations inactivating these receptorsin mouse embryos. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, andVEGFR-1 also binds VEGF-B and placenta growth factor (PlGF). VEGF-C hasbeen shown to be the ligand for Flt-4 (VEGFR-3), and also activatesVEGFR-2 (Joukov et al., 1996). A ligand for Tek/Tie-2 has been described(International Patent Application No. PCT/US95/12935 (WO 96/11269) byRegeneron Pharmaceuticals, Inc.); however, the ligand for Tie has notyet been identified.

The receptor Flt-4 is expressed in venous and lymphatic endothelia inthe fetus, and predominantly in lymphatic endothelia in the adult(Kaipainen et al., Cancer Res., 1994 54 6571-6577; Proc. Natl. Acad.Sci. USA, 1995 92 3566-3570). It has been suggested that VEGF-C may havea primary function in lymphatic endothelium, and a secondary function inangiogenesis and permeability regulation which is shared with VEGF(Joukov et al., 1996).

We have now isolated human cDNA encoding a novel protein of the vascularendothelial growth factor family. The novel protein, designated VEGF-D,has structural similarities to other members of this family.

SUMMARY OF THE INVENTION

The invention generally provides an isolated novel growth factor whichhas the ability to stimulate and/or enhance proliferation ordifferentiation of endothelial cells, isolated DNA sequences encodingthe novel growth factor, and compositions useful for diagnostic and/ortherapeutic applications.

According to one aspect, the invention provides an isolated and purifiednucleic acid molecule which encodes a novel polypeptide, designatedVEGF-D, which is structurally homologous to VEGF, VEGF-B, and VEGF-C. Ina preferred embodiment, the nucleic acid molecule is a cDNA whichcomprises the sequence set out in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6or SEQ ID NO:7. This aspect of the invention also encompasses DNAmolecules of sequence such that they hybridize under stringentconditions with DNA of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 or SEQ IDNO:7. Preferably the DNA molecule able to hybridize under stringentconditions encodes the portion of VEGF-D from amino acid residue 93 toamino acid residue 201, and isoptionally operatively linked to a DNAsequence encoding FLAG™ peptide.

Preferably, the cDNA comprises the sequence set out in SEQ ID NO:4, SEQID NO:6, or SEQ ID NO:7, more preferably that of SEQ ID NO:4.

According to a second aspect, the invention provides a polypeptidepossessing the characteristic amino acid sequence:

-   Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO:2),    said polypeptide having the ability to stimulate proliferation of    endothelial cells, and said polypeptide comprising a sequence of    amino acids substantially corresponding to the amino acid sequence    set out in SEQ ID NO:3, or a fragment or analog thereof which has    the ability to stimulate one or more of endothelial cell    proliferation, differentiation, migration or survival.

These abilities are referred to herein as “biological activities ofVEGF-D” and can readily be tested by methods known in the art.Preferably the polypeptide has the ability to stimulate endothelial cellproliferation or differentiation, including, but not limited to,proliferation or differentiation of vascular endothelial cells and/orlymphatic endothelial cells.

More preferably, the polypeptide has the sequence set out in SEQ IDNO:5, SEQ ID NO:8, or SEQ ID NO:9, and most preferably has the sequenceset out in SEQ ID NO:5.

A preferred fragment of the polypeptide invention is the portion ofVEGF-D from amino acid residue 93 to amino acid residue 201, and isoptionally linked to FLAG™ peptide. Where the fragment is linked toFLAG™, the fragment is VEGFDΔNΔC, as hereindefined.

Thus, polypeptides comprising conservative substitutions, insertions, ordeletions, but which still retain the biological activity of VEGF-D, areclearly to be understood to be within the scope of the invention. Theperson skilled in the art will be well aware of methods which canreadily be used to generate such polypeptides, for example, the use ofsite-directed mutagenesis, or specific enzymatic cleavage and ligation.The skilled person will also be aware that peptidomimetic compounds orcompounds in which one or more amino acid residues are replaced by anon-naturally occurring amino acid or an amino acid analog may retainthe required aspects of the biological activity of VEGF-D. Suchcompounds can readily be made and tested by methods known in the art,and are also within the scope of the invention.

In addition, variant forms of the VEGF-D polypeptide, which result fromalternative splicing, as are known to occur with VEGF, andnaturally-occurring allelic variants of the nucleic acid sequenceencoding VEGF-D are allencompassed within the scope of the invention.Allelic variants are well known in the art, and represent alternativeforms or a nucleic acid sequence which comprise substitution, deletionor addition of one or more nucleotides, but which do not result in anysubstantial functional alteration of the encoded polypeptide.

As used herein, the term “VEGF-D” collectively refers to thepolypeptides of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:9and fragments or analogs thereof which have the biological activity ofVEGF-D as herein defined.

Such variant forms of VEGF-D can be prepared by targeting non-essentialregions of the VEGF-D polypeptide for modification. These non-essentialregions are expected to fall outside the strongly-conserved regionsindicated in the figures herein, especially FIG. 2 and FIG. 10. Inparticular, the growth factors of the PDGF family, including VEGF, aredimeric, and VEGF-B, VEGF-C, PlGF, PDGF-A and PDGF-B show completeconservation of 8 cysteine residues in the N-terminal domains, i.e. thePDGF-like domains (Olofsson et al., 1996; Joukov et al., 1996). Thesecysteines are thought to be involved in intra- and inter-moleculardisulfide bonding. In addition, there are further strongly, but notcompletely, conserved cysteine residues in the C-terminal domains. Loops1, 2, and 3 of each subunit, which are formed by intra-moleculardisulfide bonding, are involved in binding to the receptors for thePDGF/VEGF family of growth factors (Andersson et al.: Growth Factors,1995 12 159-164). As shown herein, the cysteines conserved in previouslyknown members of the VEGF family are also conserved in VEGF-D.

The person skilled in the art thus is well aware that these cysteineresidues should be preserved in any proposed variant form, and that theactive sites present in loops 1, 2, and 3 also should be preserved.However, other regions of the molecule can be expected to be of lesserimportance for biological function, and therefore offer suitable targetsfor modification. Modified polypeptides can readily be tested for theirability to show the biological activity of VEGF-D by routine activityassay procedures such as cell proliferation tests.

It is contemplated that some modified VEGF-D polypeptides will have theability to bind to endothelial cells, i.e. to VEGF-D receptors, but willbe unable to stimulate endothelial cell proliferation, differentiation,migration, or survival. These modified polypeptides are expected to beable to act as competitive or non-competitive inhibitors of VEGF-D, andto be useful in situations where prevention or reduction of VEGF-Daction is desirable. Thus, such receptor-binding but non-mitogenic,non-differentiation inducing, non-migration inducing or non-survivalpromoting variants of VEGF-D are also within the scope of the invention,and are referred to herein as “receptor-binding but otherwise inactivevariants”.

According to a third aspect, the invention provides a purified andisolated nucleic acid encoding a polypeptide or polypeptide fragment ofthe invention. The nucleic acid may be DNA, genomic DNA, cDNA, or RNA,and may be single-stranded or double stranded. The nucleic acid may beisolated from a cell or tissue source, or of recombinant or syntheticorigin. Because of the degeneracy of the genetic code, the personskilled in the art will appreciate that many such coding sequences arepossible, where each sequence encodes the amino acid sequence shown inSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:9, an activefragment or analog thereof, or a receptor-binding but otherwise inactiveor partially inactive variant thereof.

A fourth aspect of the invention provides vectors comprising the cDNA ofthe invention or a nucleic acid according to the third aspect of theinvention, and host cells transformed or transfected with nucleic acidsor vectors of the invention. These cells are particularly suitable forexpression of the polypeptide of the invention, and include insect cellssuch as Sf9 cells, obtainable from the American Type Culture Collection(ATCC SRL-171), transformed with a baculovirus vector, and the humanembryo kidney cell line 293EBNA, transfected by a suitable expressionplasmid. Preferred vectors of the invention are expression vectors inwhich a nucleic acid according to the invention is operatively connectedto one or more appropriate promoters and/or other control sequences,such that appropriate host cells transformed or transfected with thevectors are capable of expressing the polypeptide of the invention.Other preferred vectors are those suitable for transfection of mammaliancells, or for gene therapy, such as adenovirus or retrovirus vectors orliposomes. A variety of such vectors is known in the art.

The invention also provides a method of making a vector capable ofexpressing a polypeptide encoded by a nucleic acid according to theinvention, comprising the steps of operatively connecting the nucleicacid to one or more appropriate promoters and/or other controlsequences, as described above.

The invention further provides a method of making a polypeptideaccording to the invention, comprising the steps of expressing a nucleicacid or vector of the invention in a host cell, and isolating thepolypeptide from the host cell or from the host cell's growth medium. Inone preferred embodiment of this aspect of the invention, the expressionvector further comprises a sequence encoding an affinity tag, such asFLAG™ or hexahistidine, in order to facilitate purification of thepolypeptide by affinity chromatography.

In yet a further aspect, the invention provides an antibody specificallyreactive with a polypeptide of the invention. This aspect of theinvention includes antibodies specific for the variant forms, fragmentsand analogs of VEGF-D referred to above. Such antibodies are useful asinhibitors or agonists of VEGF-D and as diagnostic agents for detectionand quantification of VEGF-D. Polyclonal or monoclonal antibodies may beused. Monoclonal and polyclonal antibodies can be raised againstpolypeptides of the invention using standard methods in the art. Forsome purposes, for example where a monoclonal antibody is to be used toinhibit effects of VEGF-D in a clinical situation, it may be desirableto use humanized or chimeric monoclonal antibodies. Methods forproducing these, including recombinant DNA methods, are also well knownin the art.

This aspect of the invention also includes an antibody which recognizesVEGF-D and which is suitably labeled.

Polypeptides or antibodies according to the invention may be labeledwith a detectable label, and utilized for diagnostic purposes.Similarly, the thus-labeled polypeptide of the invention may be used toidentify its corresponding receptor in situ. The polypeptide or antibodymay be covalently or non-covalently coupled to a suitable supermagnetic,paramagnetic, electron dense, ecogenic, or radioactive agent forimaging. For use in diagnostic assays, radioactive or non-radioactivelabels, the latter including enzyme labels or labels of thebiotin/avidin system, may be used.

Clinical applications of the invention include diagnostic applications,acceleration of angiogenesis in wound healing, tissue or organtransplantation, or to establish collateral circulation in tissueinfarction or arterial stenosis, such as coronary artery disease, andinhibition of angiogenesis in the treatment of cancer or of diabeticretinopathy. Quantitation of VEGF-D in cancer biopsy specimens may beuseful as an indicator of future metastatic risk.

Inasmuch as VEGF-D is highly expressed in the lung, and it alsoincreases vascular permeability, it is relevant to a variety of lungconditions. VEGF-D assays could be used in the diagnosis of various lungdisorders. VEGF-D could also be used in the treatment of lung disordersto improve blood circulation in the lung and/or gaseous exchange betweenthe lungs and the blood stream. Similarly, VEGF-D could be used toimprove blood circulation to the heart and O₂ gas permeability in casesof cardiac insufficiency. In like manner, VEGF-D could be used toimprove blood flow and gaseous exchange in chronic obstructive airwaydisease.

Conversely, VEGF-D antagonists (e.g., antibodies and/or inhibitors)could be used to treat conditions, such as congestive heart failure,involving accumulations of fluid in, for example, the lung resultingfrom increases in vascular permeability, by exerting an offsettingeffect on vascular permeability in order to counteract the fluidaccumulation.

VEGF-D is also expressed in the small intestine and colon, andadministrations of VEGF-D could be used to treat malabsorptive syndromesin the intestinal tract as a result of its blood circulation increasingand vascular permeability increasing activities.

Thus the invention provides a method of stimulation of angiogenesisand/or neovascularization in a mammal in need of such treatment,comprising the step of administering an effective dose of VEGF-D, or afragment or analog thereof which has the ability to stimulateendothelial cell proliferation, to the mammal.

Optionally VEGF-D may be administered together with, or in conjunctionwith, one or more of VEGF-A, VEGF-B, VEGF-C, PlGF, PDGF, FGF and/orheparin.

Conversely, the invention provides a method of inhibiting angiogenesisand/or neovascularization in a mammal in need of such treatment,comprising the step of administering an effective amount of anantagonist of VEGF-D to the mammal. The antagonist may be any agent thatprevents the action of VEGF-D, either by preventing the binding ofVEGF-D to its corresponding receptor or the target cell, or bypreventing activation of the transducer of the signal from the receptorto its cellular site of action. Suitable antagonists include, but arenot limited to, antibodies directed against VEGF-D; competitive ornon-competitive inhibitors of binding of VEGF-D to the VEGF-D receptor,such as the receptor-binding but non-mitogenic VEGF-D variants referredto above; and anti-sense nucleotide sequences complementary to at leasta part of the DNA sequence encoding VEGF-D.

The invention also provides a method of detecting VEGF-D in a biologicalsample, comprising the step of contacting the sample with a reagentcapable of binding VEGF-D, and detecting the binding. Preferably thereagent capable of binding VEGF-D is an antibody directed againstVEGF-D, more preferably a monoclonal antibody. In a preferred embodimentthe binding and/or extent of binding is detected by means of adetectable label; suitable labels are discussed above.

Where VEGF-D or an antagonist is to be used for therapeutic purposes,the dose and route of application will depend upon the condition to betreated, and will be at the discretion of the attending physician orveterinarian. Suitable routes include subcutaneous, intramuscular orintravenous injection, topical application, implants etc. Topicalapplication of VEGF-D may be used in a manner analogous to VEGF.

According to yet a further aspect, the invention providesdiagnostic/prognostic device, typically in the form of test kits. Forexample, in one embodiment of the invention there is provided adiagnostic/prognostic test kit comprising an antibody to VEGF-D andmeans for detecting, and more preferably evaluating, binding between theantibody and VEGF-D. In one preferred embodiment of thediagnostic/prognostic device according to the invention, either theantibody or the VEGF-D is labeled with a detectable label, and eitherthe antibody or the VEGF-D is substrate-bound, such that theVEGF-D-antibody interaction can be established by determining the amountof label attached to the substrate following binding between theantibody and the VEGF-D. In a particularly preferred embodiment of theinvention, the diagnostic/prognostic device may be provided as aconventional ELISA kit.

In another alternative embodiment, the diagnostic/prognostic device maycomprise polymerase chain reaction means for establishing the genomicsequence structure of a VEGF-D gene of a test individual, and comparingthis sequence structure with that disclosed in this application in orderto detect any abnormalities, with a view to establishing whether anyaberrations in VEGF-D expression are related to a given diseasecondition.

In accordance with a further aspect, the invention relates to a methodof detecting aberrations in VEGF-D gene structure in a test subjectwhich may be associated with a disease condition in said test subject.This method comprises providing a DNA sample from said test subject;contacting the DNA sample with a set of primers specific to VEGF-D DNAoperatively coupled to a polymerase; selectively amplifying VEGF-D DNAfrom the sample by polymerase chain reaction; and comparing thenucleotide sequence of the amplified VEGF-D DNA from the sample with thenucleotide sequences set forth in SEQ ID NO:1 or SEQ ID NO:4. Theinvention also includes the provision of a test kit comprising a pair ofprimers specific to VEGF-D DNA operatively coupled to a polymerase,whereby said polymerase is enabled to selectively amplify VEGF-D DNAfrom a DNA sample.

Another aspect of the invention concerns the provision of apharmaceutical composition comprising either VEGF-D polypeptide or afragment or analog thereof which promotes proliferation of endothelialcells, or an antibody thereto. Compositions which comprise VEGF-Dpolypeptide may optionally further comprise one or more of VEGF, VEGF-B,VEGF-C, and/or heparin.

In another aspect, the invention relates to a protein dimer comprisingVEGF-D polypeptide, particularly a disulfide-linked dimer. The proteindimers of the invention include both homodimers of VEGF-D polypeptideand heterodimers of VEGF-D and VEGF, VEGF-B, VEGF-C, PlGF, or PDGF.

According to a yet further aspect of the invention there is provided amethod for isolation of VEGF-D comprising the step of exposing a cellwhich expresses VEGF-D to heparin to facilitate release of VEGF-D fromthe cell, and purifying the thus-released VEGF-D.

Another aspect of the invention involves providing a vector comprisingan anti-sense nucleotide sequence which is complementary to at least apart of a DNA sequence which encodes VEGF-D or a fragment or analogthereof which promotes proliferation of endothelial cells. According toa yet further aspect of the invention, such a vector comprising ananti-sense sequence may be used to inhibit, or at least mitigate, VEGF-Dexpression. The use of a vector of this type to inhibit VEGF-Dexpression is favored in instances where VEGF-D expression is associatedwith a disease, for example, where tumors produce VEGF-D in order toprovide for angiogenesis. Transformation of such tumor cells with avector containing an anti-sense nucleotide sequence would suppress orretard angiogenesis, and so would inhibit or retard growth of the tumor.

Polynucleotides of the invention such as those described above,fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridize thereto under stringent conditionsall are useful for identifying, purifying, and isolating polynucleotidesencoding other, non-human, mammalian forms of VEGF-D. Thus, suchpolynucleotide fragments and variants are intended as aspects of theinvention. Exemplary stringent hybridization conditions are as follows:hybridization at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8, 50% formamide;and washing at 42° C. in 0.2×SSC. Those skilled in the art understandthat it is desirable to vary these conditions empirically based on thelength and the GC nucleotide base content of the sequences to behybridized, and that formulae for determining such variation exist. See,for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual”,Second Edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory(1989).

Moreover, purified and isolated polynucleotides encoding other,non-human, mammalian VEGF-D forms also are aspects of the invention, asare the polypeptides encoded thereby, and antibodies that arespecifically immunoreactive with the non-human VEGF-D variants. Thus,the invention includes a purified and isolated mammalian VEGF-Dpolypeptide, and also a purified and isolated polynucleotide encodingsuch a polypeptide.

It will be clearly understood that nucleic acids and polypeptides of theinvention may be prepared by synthetic means or by recombinant means, ormay be purified from natural sources.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show a comparison between the sequences of human VEGF-D andhuman VEGF₁₆₅ (FIG. 1A), human VEGF-B (FIG. 1B), human VEGF-C (FIG. 1C)and human PlGF (FIG. 1D). The box indicates residues which match thosein human VEGF-D exactly.

FIG. 2 shows sequence alignments between the sequences of human VEGF-D,human VEGF₁₆₅, human VEGF-B, human VEGF-C and human PlGF. The boxesindicate residues that match the VEGF-D sequence exactly.

FIG. 3 shows the amino acid sequence of human VEGF-D (SEQ ID NO:3), aspredicted from the cDNA sequence (SEQ ID NO:1). The boxes indicatepotential sites for N-linked glycosylation.

FIG. 4 shows the nucleotide sequence of a second cDNA sequence encodinghuman VEGF-D (SEQ ID NO:4), isolated by hybridization from a commercialhuman lung cDNA library; this cDNA contains the entire coding region forhuman VEGF-D.

FIG. 5 shows the amino acid sequence for human VEGF-D (SEQ ID NO:5)deduced from the sequence of the cDNA of FIG. 4.

FIG. 6 shows the nucleotide sequence of cDNA encoding mouse VEGF-D1 (SEQID NO:6), isolated by hybridization screening for acommercially-available mouse lung cDNA library.

FIG. 7 shows the nucleotide sequence of cDNA encoding mouse VEGF-D2 (SEQID NO:7), isolated from the same library as in FIG. 6.

FIG. 8 shows the deduced amino acid sequences for mouse VEGF-D1 (SEQ IDNO:8) and VEGF-D2 (SEQ ID NO:9).

FIG. 9 shows a comparison between the deduced amino acid sequences ofmouse VEGF-D1, mouse VEGF-D2, and human VEGF-D.

FIG. 10 shows sequence alignments between the amino acid sequences ofhuman VEGF-D, human VEGF₁₆₅, human VEGF-B, human VEGF-C, and human PlGF.

FIG. 11 shows the results of a bioassay in which conditioned medium fromCOS cells expressing either VEGF-A or VEGF-L was tested for ability tobind to the extracellular domain of a chimeric receptor expressed inBa/F3 cells.

FIGS. 12A-12B show the results of immunoprecipitation and Westernblotting analysis of VEGF-D peptides.

(A) pEFBOSVEGFDfullFLAG and pcDNA-1VEGF-A were transfected into COScells and biosynthetically labeled with ³⁵S-cysteine/methionine for 4hours. The supernatants from these cultures were immunoprecipitated witheither M2 gel or an antiserum directed to VEGF-A coupled to proteinA.Washed beads were eluted with an equal volume of 2×SDS-PAGE samplebuffer and boiled. The samples were then resolved by 12% SDS-PAGE. Lanesmarked with an asterix (*) indicate where samples were reduced withdithiothreitol and alkylated with iodoacetamide. Molecular weightmarkers are indicated. fA and fB indicate the 43 kD and 25 kD speciesimmunoprecipitated by the M2 gel from the COS cells expressingpEFBOSVEGFDfullFLAG.

(B) Western blotting analysis of purified VEGFDΔNΔC. An aliquot ofmaterial eluted from the M2 affinity column (fraction #3, VEGFDΔNΔC) wascombined with 2×SDS-PAGE sample buffer and resolved on a 15% SDS-PAGEgel. The proteins were then transferred to nitrocellulose membrane andprobed with either monoclonal antibody M2 or a control isotype-matchedantibody (Neg). Blots were developed using a goat anti-mouse-HRPsecondary antibody and chemiluminescence (ECL, Amersham). MonomericVEGFDΔNΔC is arrowed, as is the putative dimeric form of this peptide(VEGFDΔNΔC″). Molecular weight markers are indicated.

FIG. 13 shows the results of analysis of VEGFDΔNΔC protein using theVEGFR2 bioassay. Recombinant VEGFDΔNΔC, and material purified by M2affinity chromatography, was assessed using the VEGFR2 bioassay.Bioassay cells (10⁴), washed to remove IL-3, were incubated withaliquots of conditioned medium from VEGF-D transfected COS cells,fraction #1 from the affinity column (void volume), or fraction #3 fromthe affinity column (containing VEGFDΔNΔC). All samples were tested atan initial concentration of 20% (i.e., ⅕) followed by doublingdilutions. Cells were allowed to incubate for 48 hours at 37° C. in ahumidified atmosphere of 10% CO₂. Cell proliferation was quantitated bythe addition of 1 μCi of ³H-thymidine and counting the amountincorporated over a period of 4 hours.

FIG. 14 shows stimulation of tyrosine phosphorylation of the VEGFR3receptor (Flt-4) on NIH3T3 cells by culture supernatant from HF cellsinfected with a recombinant baculovirus vector transformed with VEGF-D.

FIG. 15 shows stimulation of tyrosine phosphorylation of the VEGFR2receptor (KDR) in PAE cells by culture supernatant prepared as in FIG.14.

FIG. 16 shows the mitogenic effect of VEGFDΔNΔC on bovine aorticendothelial cells (BAEs). BAEs were treated with fraction #3 containingVEGFDΔNΔC and, as positive control, purified VEGF-A as described in thetext. The result obtained using medium without added growth factor isdenoted Medium Control.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by reference to thefigures, and to the following non-limiting examples.

Example 1

It has been speculated that no further members of the VEGF family willbe found, because there are no known orphan receptors in the VEGFRfamily. Furthermore, we are not aware of any suggestion in the prior artthat other such family members would exist.

A computer search of nucleic acid databases was carried out incidentallyto another project, using as search topics the amino acid sequences ofVEGF, VEGF-B, VEGF-C, and PlGF. Several cDNA sequences were identifiedby this search. One of these sequences, GenBank Accession No. H24828,encoded a polypeptide which was similar in structure to thecysteine-riched C-terminal region of VEGF-C. This sequence was obtainedfrom the database of expressed sequence tags (dbEST), and for thepurposes of this specification is designated XPT. The XPT cDNA had beenisolated from a human cDNA library designated “Soares Breast 3NbHBst”,which was constructed using mRNA from an adult human female breasttissue. As far as can be ascertained, this sample was normal breasttissue. Sequencing of the XPT DNA was performed pursuant to theIntegrated Molecular Analysis of Genome Expression Consortium (IMAGEConsortium), which solicits cDNA libraries from laboratories around theworld, arrays the cDNA clones, and provides them to other organizationsfor sequencing.

The XPT sequence shown in the database was 419 nucleotides long, andencoded an amino acid sequence similar to the C-terminal 100 amino acidsof VEGF-C, i.e., approximately residues 250 to 350, using the numberingsystem of Joukov et al. (1996). Similarly, cysteine-rich regions arefound in other proteins, which are entirely unrelated in function to theVEGF family, for example, the secreted silk-like protein sp185synthesized in the salivary glands of the midge Chironomus tentans. Thisprotein is encoded by the gene BR3, located in a Balbiani ring, a tissuespecific chromosome “puff” found on polytene chromosomes in the midgesalivary gland (Dignam and Case: Gene, 1990 88 133-140; Paulsson et al.,J. Mol. Biol., 1990 211 331-349). It is stated in Joukov et al. (1996)that the sp185-like structural motif in VEGF-C may fold into anindependent domain, which is thought to be at least partially cleavedoff after biosynthesis, and that there is at least one cysteine motif ofthe sp185 type in the C-terminal region of VEGF.

FIG. 3 of Joukov et al. shows that the last two-thirds of the C-terminalcysteine-rich region of VEGF-C do not align with VEGF or PlGF, and infact could be considered a C-terminal extension of VEGF-C which is notpresent in VEGF or PlGF. The sequence encoded by XPT is similar to thisextension. As the XPT cDNA was truncated at its 5′ end, it was notpossible to deduce or predict any amino acid sequence for regionsN-terminal to the cysteine-rich domain. Thus the portion of VEGF-C whichis similar to the XPT-derived sequence does not extend to regions ofVEGF-C which are conserved among other members of the VEGF family.

As described above, it was not possible to predict whether theN-terminal region of the polypeptide encoded by a full-length XPTnucleic acid (as distinct from the truncated XPT cDNA reported in dbEST)would show any further homology to any member of the VEGF family, inparticular VEGF-C, which has a further N-terminal 250 amino acids. Forexample, the naturally-occurring protein encoded by a full-length XPTnucleic acid could have been the human homolog of the midge salivarygland protein. Alternatively, the type of cysteine-rich motif encoded bytruncated XPT cDNA could be widely distributed among proteins, as aremany structural domains. For example, clusters of cysteine residues maybe involved in metal binding, formation of intramolecular disulfidebonds to promote accurate protein folding, or formation ofintermolecular disulfide bonds for assembly of protein subunits intocomplexes (Dignam and Chase, 1990). In order to determine whether thetruncated XPT cDNA was derived from sequences encoding a VEGF-relatedmolecule, it was necessary to isolate a much longer cDNA.

Example 2 Cloning of cDNA Encoding VEGF-D

A sample of the XPT cDNA reported in dbEST was obtained from theAmerican Type Culture Collection, which is a registered supplier of cDNAclones obtained by the IMAGE Consortium. The identity of the XPT cDNAwas confirmed by nucleotide sequencing, using the dideoxy chaintermination method (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977 745463-5467).

The XPT cDNA was used as a hybridization probe to screen a human breastcDNA library, which was obtained commercially from Clontech. Onepositive clone was isolated, and this clone was then sequenced on bothstrands. The nucleotide sequence was compiled, and an open reading framewas identified. The nucleic acid sequence is set out in SEQ ID NO:1. Thepolypeptide encoded by this sequence was designated VEGF-D, and itsdeduced amino acid sequence, designated SEQ ID NO:3, is set out in FIG.3. In FIG. 3, putative sites of N-linked glycosylation, with theconsensus sequence N—X—S/T in which X is any amino acid, are indicatedby the boxes.

Example 3 Characteristics of VEGF-D

The amino acid sequence of VEGF-D was compared with those of humanVEGF-A₁₆₅, VEGF-B, VEGF-C, and PlGF. These comparisons are set out inFIGS. 1A to D, respectively. The degree of sequence homology wascalculated, and if gaps in sequence introduced for the purposes ofalignment are not considered in the calculation, VEGF-D is 31% identicalto VEGF, 48% identical to VEGF-C, 28% identical to VEGF-B, and 32%identical to PlGF. Thus, the most closely-related protein identified wasVEGF-C.

Computer searches of the GenBank, EMBL and SwissProt nucleic aciddatabases did not reveal any protein sequences identical to VEGF-D. Asexpected from the sequence alignment referred to above, the most closelyrelated protein found in these databases was VEGF-C. Searches of dbESTwere also performed, but did not reveal any sequences encompassing theentire coding region of VEGF-D. The sequence of VEGF-D is unrelated tothat of Tie-2 ligand 1 as disclosed in WO 96/11269.

It is important to bear in mind that the only homologies detected wereat the level of the amino acid sequence. Thus, it would not have beenpossible to isolate the cDNA or gDNA encoding VEGF-D by methods such aslow-stringency hybridization with a nucleic acid sequence encodinganother member of the VEGF family.

VEGF-D appears to be most closely related to VEGF-C of all the membersof the VEGF family. Because the VEGF-D amino acid sequence includes thecysteine-rich sp185-like motif which is found in VEGF-C, the polypeptideof the invention may play an important functional role in lymphaticendothelia. While we do not wish to be bound by any proposed mechanism,it is thought that VEGF-C and VEGF-D may constitute a silk-like matrixover which endothelial cells can grow. Lymphatic vessels have nobasement membrane, so the silk-like matrix can form a basementmembrane-like material. This may be important in promoting cell growthand/or in cell differentiation, and may be relevant to cancer,especially metastasis, drug therapy, cancer prognosis, etc.

Example 4 Biological Characteristics of VEGF-D

The cDNA sequence of VEGF-D was used to predict the deduced amino acidsequence of VEGF-D, the biochemical characteristics of the encodedpolypeptide, including the numbers of strongly basic, strongly acidic,hydrophobic and polar amino acids, the molecular weight, the isoelectricpoint, the charge at pH 7, and the compositional analysis of the wholeprotein. This analysis was performed using the Protean protein analysisprogram, Version 1.20 (DATASTAR). These results are summarized in Tables1 and 2 below. Table 1 also shows the codon usage.

TABLE 1 Translated DNA Sequence of VEGF-D contig x(1,978) With StandardGenetic Code Molecular Weight 37056.60 Daltons 425 Amino Acids 46 StrongBasic(+) Amino Acids (K, R) 41 Strong Acidic(−) Amino Acids (D, E) 79hydrophobic Amino Acids (A, I, L, F, W, V) 108 Polar Amino Acids (N, C,Q, S, T, Y) 7.792 Isoelectric Point 6.371 Charge at pH 7.0 Total numberof bases translated is 978 % A = 28.73 [281] % G = 23.11 [226] % T =23.21 [227] % C = 24.95 [244] % Ambiguous = 0.00 [0] % A + T = 51.94[508] % C + G = 48.06 [470] Davis, Botstein, Roth Melting Temp ° C.84.09 Wallace Temp ° C. 3384.00 Codon usage: ccg ( ) 0 # ugc Cys (CS 14# cuc Leu (L) 6 # ucg Ser (S) uaa ( ) 0 # ugu Cys (C) 16 # cug Leu (L) 4# ucu Ser (S) uag ( ) 0 # ——— Cys (C) 30 # cuu Leu (L) 2 # ——— Ser (S) 3——— ( ) 0 # caa Gln (Q) 1 # uua Leu (L) 1 # uga Ter (.) gca Ala (A) 5 #cag Gln (Q) 11 # uug Leu (L) 5 # ——— Ter (.) gcc Ala (A) 4 # ——— Gln (Q)12 # ——— Leu (L) 23 # aca Thr (T) gcg Ala (A) 1 # gaa Glu (E) 16 # aaaLys (K) 13 # acc Thr (T) gcu Ala (A) 5 # gag Glu (E) 12 # aag Lys (K) 10# acg Thr (T) ——— Ala (A) 15 # ——— Glu(E) 28 # ——— Lys (K) 23 # acu Thr(T) aga Arg (R) 7 # gga Gly(G) 1 # aug Met (M) 6 # ——— Thr (T) 2 agg Arg(R) 5 # ggc Gly (G) 2 # ——— Met (M) 6 # ugg Trp (W) cga Arg (R) 5 # gggGly(G) 3 # uuc Phe (F) 4 # ——— Trp (W) cgc Arg (R) 4 # ggu Gly (G) 2 #uuu Phe (F) 8 # uac Tyr (Y) cgg Arg (R) 1 # ——— Gly (G) 8 # ——— Phe (F)12 # uau Tyr (Y) cgu Arg (R) 1 # cac His (H) 7 # cca Pro (P) 9 # ——— Tyr(Y) ——— Arg (R) 23 # cau His (H) 7 # ccc Pro (P) 6 # gua Val (V) aac Asn(N) 5 # ——— His (H) 14 # ccu Pro (P) 8 # guc Val (V) aau Asn (N) 4 # auaIle (I) 2 # ——— Pro (P) 23 # gug Val (V) ——— Asn (N) 9 # auc Ile (I) 6 #agc Ser (S) 6 # guu Val (V) gac Asp (D) 8 # auu Ile (I) 5 # agu Ser (S)8 # ——— Val (V) 1 gau Asp (D) 5 # ——— Ile (I) 13 # uca Ser (S) 5 # nnn??? (X) gau Asp (D) 5 # ——— Ile (I) 13 # uca Ser (S) 5 # nnn ??? (X) ———Asp (D) 13 # cua Leu (L) 5 # ucc Ser (S) 7 # TOTAL 32 Contig 2: ContigLength: 2379 bases Average Length/Sequence:  354 bases Total SequenceLength: 4969 bases

TABLE 2 Predicted Structural Class of the Whole Protein: Deléage & RouxModification of Nishikawa & Ooi 1987 Analysis Whole Protein MolecularWeight 37056.60 m.w. Length 325 1 microgram = 26.986 pMoles MolarExtinction coefficient 30200 ± 5% 1 A(280) = 1.23 mg/ml IsoelectricPoint 7.79 Charge at pH 7 6.37 Whole Protein Composition Analysis AminoAcid(s) Number count % by weight % by frequency Charged (RKHYCDE) 13446.30 41.23 Acidic (DE) 41 13.79 12.62 Basic (KR) 46 17.65 14.15 Polar(NCQSTY) 108 30.08 33.23 Hydrophobic (AILFWV) 79 23.86 24.31 A Ala 152.88 4.62 C Cys 30 8.35 9.23 D Asp 13 4.04 4.00 E Glu 28 9.75 8.62 F Phe12 4.77 3.69 G Gly 8 1.23 2.46 H His 14 5.18 4.31 I Ile 13 3.97 4.00 KLys 23 7.96 7.08 L Leu 23 7.03 7.08 M Met 6 2.12 1.85 N Asn 9 2.77 2.77P Pro 23 6.08 7.08 Q Gln 12 4.15 3.69 R Arg 23 9.69 7.08 S Ser 33 7.7610.15 T Thr 21 5.73 6.46 V Val 12 3.21 3.69 W Trp 4 2.01 1.23 Y Trp 31.32 0.92 B Asx 0 0.00 0.00 Z Glx 0 0.00 0.00 X Xxx 0 0.00 0.00 . Ter 00.00 0.00

This analysis predicts a molecular weight for the unprocessed VEGF-Dmonomer of 37 kilodaltons (kD), compared to the experimentallydetermined values (for the fully processes peptides) of 20 to 27 kD forVEGF-A monomers, 21 kD for the VEGF-B monomer and 23 kD for the VEGF-Cmonomer.

Example 5

The original isolation of a cDNA for VEGF-D, described in Example 2involved hybridization screening of a human breast cDNA library. As onlyone cDNA clone for VEGF-D was thus isolated, it was not possible toconfirm the structure of the cDNA by comparison with other independentlyisolated VEGF-D cDNAs. The work described in this example, whichinvolved isolation of additional human VEGF-D cDNA clones, was carriedout in order to confirm the structure of human VEGF-D cDNA. In addition,mouse VEGF-D cDNA clones were isolated.

Two cDNA libraries which had been obtained commercially from Stratagene,one for human lung and one for mouse lung (catalogue numbers 937210 and936307, respectively) were used for hybridization screening with aVEGF-D cDNA probe. The probe, which spanned from nucleotides 1817 to2495 of the cDNA for human VEGF-D described in Example 2, was generatedby polymerase chain reaction (PCR) using a plasmid containing the VEGF-DcDNA as template and the following two oligonucleotides:

(SEQ ID NO: 10) 5′-GGGCTGCTTCTAGTTTGGAG, and (SEQ ID NO: 11)5′-CACTCGCAACGATCTTCGTC.

Approximately two million recombinant bacteriophage were screened withthis probe from each of the two cDNA libraries. Nine human and six mousecDNA clones for VEGF-D were subsequently isolated.

Two of the nine human cDNA clones for VEGF-D were sequenced completelyusing the dideoxy chain termination method (Sanger et al. Proc. Natl.Acad. Sci. USA, 1977 74 5463-5467). The two cDNAs contained the entirecoding region for human VEGF-D, and were identical except that one ofthe clones was five nucleotides longer than the other at the5′-terminus. The nucleotide sequence of the shorter cDNA is shown inFIG. 4, and is designated SEQ ID NO:4. The amino acid sequence for humanVEGF-D (hVEGF-D) deduced from this cDNA was 354 residues long, and isshown in FIG. 5; this is designated SEQ ID NO:5. The sequences of the 5′regions of five of the other human VEGF-D cDNA clones were alsodetermined. For each clone, the sequence that was characterizedcontained more than 100 nucleotides of DNA immediately downstream fromthe translation start site of the coding region. In all cases, thesequences of these regions were identical to corresponding regions ofthe human VEGF-D cDNA shown in FIG. 4.

All six mouse cDNA clones for VEGF-D were sequenced completely. Only twoof the clones contained an entire coding region for VEGF-D; the otherclones were truncated. The nucleotide sequences of the two clones withan entire coding region are different, and encode amino acid sequencesof different sizes. The longer amino acid sequence is designatedmVEGF-D1, and the shorter sequence is designated mVEGF-D2. Thenucleotide sequences of the cDNAs encoding mVEGF-D1 and mVEGF-D2 areshown in FIGS. 6 and 7 respectively. The deduced amino acid sequencesfor mVEGF-D1 and mVEGF-D2 are shown in FIG. 8. These sequences arerespectively designated SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQID NO: 9. The differences between the amino acid sequences are:

i) an insertion of five amino acids (DFSFE) after residue 30 in mVEGF-D1in comparison to mVEGF-D2;

ii) complete divergence of the C-terminal ends after residue 317 inmVEGF-D1 and residue 312 in mVEGF-D2, which results in mVEGF-D1 beingconsiderably longer.

Three of the four truncated cDNAs for mouse VEGF-D encoded theC-terminal region, but not the N-terminal 50 amino acids. All three ofthese cDNAs encoded a C-terminal end for VEGF-D which is identical tothat for mVEGF-D2. The other truncated cDNA encoded only the N-terminalhalf of VEGF-D. The amino acid sequence deduced from this cDNA containedthe five amino acids DFSFE immediately after residue 30 found inmVEGF-D1, but not in mVEGF-D2.

As described above, the entire sequence of the human VEGF-D cDNA clonereported in this example has been validated by comparison with that fora second human clone. In addition, the sequence of the 5′ end of thecoding region was found to be identical in five other human VEGF-D cDNAclones. In contrast, the sequence reported in Example 2 contained mostof the coding region for VEGF-D, but was incorrect near the 5′-end ofthis region. This was probably because the VEGF-D cDNA was truncatednear the 5′-end of the coding region and at that point had been ligatedwith another unidentified cDNA, and consequently the first 30 codons ofthe true coding sequence for VEGF-D had been deleted and replaced with amethionine residue. This methionine residue was defined as theN-terminal amino acid of the VEGF-D sequence presented in Example 2.

The N-terminal regions of the deduced amino acid sequences of mouseVEGF-D1 and VEGF-D2 are very similar to that deduced for human VEGF-D(see FIG. 9). This also indicates that the correct deduced amino acidsequence for human VEGF-D is reported in this example. The N-terminal 25amino acids of human VEGF-D form an extremely hydrophobic region, whichis consistent with the notion that part of this region may be a signalsequence for protein secretion. FIG. 10 shows the alignment of the humanVEGF-D sequence with the sequences of other members of the VEGF familyof growth factors, namely human VEGF₁₆₅ (hVEGF₁₆₅), human VEGF-B(hVEGF-B), human VEGF-C (hVEGF-C), and human Placental Growth Factor(hPlGF). When gaps in the alignments are ignored for the purposes ofcalculation, human VEGF-D is found to be 31% identical in amino acidsequence to human VEGF₁₆₅, 28% identical to human VEGF-B, 48% identicalto VEGF-C and 32% identical to human PlGF. Clearly, VEGF-C is the memberof this family which is most closely-related to VEGF-D.

The differences in sequence for mouse VEGF-D1 and VEGF-D2 most probablyarise from differential mRNA splicing. The C-terminal 41 amino acidresidues of VEGF-D1 are deleted in VEGF-D2, and are replaced with 9residues which are not closely related to the VEGF-D1 sequence.Therefore, 4 cysteine residues present near the C-terminus of VEGF-D1are deleted in VEGF-D2. This change may alter the tertiary or quaternarystructures of the protein, or may affect the localization of the proteinin the cell or the extracellular environment. The C-terminal end ofhuman VEGF-D resembles that of mouse VEGF-D1, not mouse VEGF-D2. Thesmall 5 amino acid insertion after residue 30 in mouse VEGF-D1, which isnot present in either mouse VEGF-D2 or human VEGF-D, may influenceproteolytic processing of the protein.

VEGF-D is highly conserved between mouse and man. Eighty-five percent ofthe amino acid residues of human VEGF-D are identical in mouse VEGF-D1.This is likely to reflect conservation of protein function. Putativefunctions for VEGF-D have been proposed herein. Although we have notfound alternative forms of human VEGF-D cDNA, it is possible that theRNA splice variation which gives rise to numerous forms of mRNA formouse VEGF-D may also occur in human tissues.

Example 6 Expression of VEGF-D in COS Cells

A fragment of the human cDNA for VEGF-D, spanning from nucleotide 1 to1520 of the sequence shown in FIG. 4 and containing the entire codingregion, was inserted into the mammalian expression vector pcDNA1-amp.The vector was used to transiently transfect COS cells by theDEAE-Dextran method as described previously (Aruffo and Seed, 1987) andthe resulting conditioned cell culture media, collected after 7 days ofincubation, were concentrated using Amicon concentrators (Centricon 10with a 10,000 molecular weight cut off) according to the manufacturer.The plasmids used for transfections were the expression construct forhuman VEGF-D and, as positive control, a construct made by insertion ofmouse VEGF-A cDNA into pcDNA1-amp. The conditioned media were tested intwo different bioassays, as described below, and the results demonstratethat the COS cells did, in fact, express and secrete biologically-activeVEGF-D.

Example 7 Bioassay for Capacity of VEGF-D to Bind to VEGF Receptor-2

As shown in Example 5, VEGF-D is closely related in primary structure toother members of the VEGF family. Most members of this protein familyare mitogenic and/or chemotactic for endothelial cells (Keck et al.,1989; Leunq et al., 1989; Joukov, et al., 1996; Olofsson et al., 1996).In addition, VEGF-A (previously known as VEGF), the first member of theVEGF family to be described in the literature, is a potent inducer ofvascular permeability (Keck et al., 1989). As protein structure is animportant determinant of protein function, it seemed likely that VEGF-Dmight also be mitogenic for endothelial cells or induce vascularpermeability. Therefore human VEGF-D was tested in a bioassay for itscapacity to bind to VEGF receptor-2 (VEGFR2; also known as Flk-1), anendothelial cell-specific receptor which, when activated by VEGF-A, isthought to give rise to a mitogenic signal (Strawn et al., 1996).

A bioassay for detection of growth factors which bind to VEGFR2 has beendeveloped in the factor-dependent cell line Ba/F3, and is described inour earlier patent application, No. PCT/US95/16755. These cells grow inthe presence of interleukin-3 (IL-3); however, removal of this factorresults in cell death within 48 hours. If another receptor capable ofdelivering a growth stimulus is transfected into the Ba/F3 cells, thecells can be rescued by the specific growth factor which activates thatreceptor when the cells are grown in medium lacking IL-3. In thespecific case of receptor-type tyrosine kinases (e.g., VEGFR2), chimericreceptors containing the extracellular domain of the receptor tyrosinekinase and the transmembrane and cytoplasmic domains of theerythropoietin receptor (EpoR) can be utilized. In this case stimulationwith the ligand (e.g., VEGF), which binds to the extracellular domain ofthe chimeric receptor, results in signalling via the EpoR cytoplasmicdomain and subsequent rescue of the cell line in growth medium lackingTL-3. The construction of the chimeric receptor used in this study,consisting of the mouse VEGFR2 extracellular domain and the mouse EpoRtransmembrane and cytoplasmic domains, and the bioassay itself, aredescribed below.

Plasmid Construction i) Construction of a Plasmid for GeneratingChimeric VEGFR2 Receptors

To obtain a plasmid construct with which DNA encoding the extracellulardomain of mouse VEGFR2 could easily be ligated with DNA encoding otherprotein domains, site-directed mutagenesis was used to generate a BglIIrestriction enzyme site at the position of mouse VEGFR2 cDNA whichencoded the junction of the extracellular domain and the transmembranedomain. The full-length clone of the mouse VEGFR2 cDNA described byOelrichs et al. (1993) was subcloned into the mammalian expressionvector pcDNA1-amp, using the BstXI restriction enzyme site. Singlestranded UTP+DNA was generated using the M13 origin of replication, andthis was used as a template to generate mouse VEGFR2 cDNA containing theBglII site at the desired position. The plasmid containing the alteredVEGFR2 cDNA was designated pVEGFR2Bgl. DNA fragments encoding thetransmembrane and cytoplasmic domains of any receptor can be inserted atthe BglII site of pVEGFR2Bgl in order to generate chimeric VEGFR2receptors.

Ii) Construction of VEGFR2/EpoR Chimeric Receptor

The mouse EpoR cDNA was subcloned into the expression vector pcDNA1-amp,and single stranded DNA was generated as a template for mutagenesis. ABglII restriction enzyme site was inserted into the EpoR cDNA at theposition encoding the junction of the transmembrane and extracellulardomains of the EpoR to allow direct ligation of this DNA fragment to themodified cDNA encoding the extracellular domain of VEGFR2 in pVEGFR2Bgl.In addition, a BglII site in the cytoplasmic domain of the EpoR wasremoved by a silent single nucleotide substitution. The DNA fragmentencoding the transmembrane and cytoplasmic domains of EpoR was then usedto replace the portion of pVEGFR2Bgl encoding the transmembrane andcytoplasmic domains of VEGFR2. Thus a single reading frame was generatedwhich encoded the chimeric receptor consisting of the VEGFR2extracellular domain and the EpoR transmembrane and cytoplasmic domains.

The DNA fragment encoding the chimeric receptor was subcloned into theexpression vector pBOS, and co-transfected into the Ba/F3 cell line withplasmid pgk-neo at a ratio of 1:20. Cells expressing the VEGFR2-EpoRprotein were selected by flow cytometry analysis using a monoclonalantibody to the VEGFR2 extracellular domain (MAb 4H3). This monoclonalantibody is described in Australian Patent Application No. PM 3794,filed 10 Feb. 1994. Cell lines expressing higher levels of VEGFR2-EpoRwere selected by growing the cells in 5 μg/ml MAb 4H3 or 25 ng/ml ofrecombinant VEGF. A cell line expressing high levels of VEGFR2-EpoR,designated Ba/F3-NYK-EpoR, was used for the bioassay.

The Bioassay

The Ba/F3-NYK-EpoR cells described above were washed three times in PBSto remove all IL-3 and resuspended at a concentration of 1000 cells per13.5 μl of culture medium and 13.5 μl was aliquoted per well of a60-well Terasaki plate. Conditioned media from transfected COS cellswere then diluted into the cell culture medium. Cells expressing achimeric receptor consisting of the extracellular domain of theendothelial cell receptor Tie2 and the transmembrane and cytoplasmicdomains of EpoR were used as a non-responding control cell line. Cellswere incubated for 48-96 hours, during which the cells incubated in cellculture medium alone had died and the relative survival/proliferationseen in the other wells (i.e., in the presence of COS cell-conditionedmedia) was scored by counting the viable cells present per well.

The conditioned medium from COS cells which had been transientlytransfected with expression plasmids was concentrated 30-fold and usedin the VEGFR2 bioassay. Concentrated conditioned medium from COS cellstransfected with pcDNA1-amp was used as negative control.

The results are shown in FIG. 11, with the percentage of 30-foldconcentrated COS cell-conditioned medium in the incubation medium(vol/vol) plotted versus the number of viable cells in the well after 48hours of incubation. Clearly, the conditioned medium containing eitherVEGF-A or VEGF-D was capable of promoting cell survival in this assay,indicating that both proteins can bind to and activate VEGFR2.

Example 8 Vascular Permeability Assay

Human VEGF-D, prepared as in Example 6 and concentrated 30-fold, wastested in the Miles vascular permeability assay (Miles and Miles, 1952)performed in anaesthetized guinea pigs (albino/white, 300-400 g).Concentrated conditioned medium for COS cells transfected withpcDNA1-amp was again used as a negative control. Guinea pigs wereanaesthetized with chloral-hydrate (3.6 g/100 ml; 0.1 ml per 10 g ofbody weight). The backs of the animals were then carefully shaved withclippers. Animals were given an intracardiac injection of Evans Blue dye(0.5% in MT PBS, 0.5 ml) using a 23G needle, and were then injectedintra-dermally with 100-150 μl of concentrated COS cell-conditionedmedium. After 15-20 min the animals were sacrificed and the layer ofskin on the back excised to expose the underlying blood vessels. Forquantitation, the area of each injection was excised and heated to 45°C. in 2-5 ml of formamide. The resulting supernatants, containingextravasated dye, were then examined spectrophotometrically at 620 nm.

For animal 1, the absorbance at 620 nm arising from injection of 30-foldconcentrated VEGF-A conditioned medium was 0.178, that for the 30-foldconcentrated VEGF-D conditioned medium was 0.114, and that for 30-foldconcentrated medium from cells transfected with pcDNA1-amp was 0.004.For animal 2, the 30-fold concentrated media were diluted 4-fold in cellculture medium before intra-dermal injection. The absorbance at 620 nmfor the VEGF-A conditioned sample was 0.141, that for the VEGF-Dconditioned sample was 0.116, and that for a sample matched for serumcontent as negative control was 0.017. The enhanced extravasation of dyeobserved for both animals in the presence of VEGF-A or VEGF-Ddemonstrated that both of these proteins strongly induced vascularpermeability.

The data described here indicate that VEGF-D is a secreted proteinwhich, like VEGF-A, binds to and activates VEGFR2 and can inducevascular permeability.

Example 9 Bioactivities of Internal VEGF-D Polypeptides

The deduced amino acid sequence for VEGF-D includes a central regionwhich is similar in sequence to all other members of the VEGF family(approximately residues 101 to 196 of the human VEGF-D amino acidsequence as shown in the alignment in FIG. 10). Therefore, it wasthought that the bioactive portion of VEGF-D might reside in theconserved region. In order to test this hypothesis, the biosynthesis ofVEGF-D was studied, and the conserved region of human VEGF-D wasexpressed in mammalian cells, purified, and tested in bioassays asdescribed below.

Plasmid Construction

A DNA fragment encoding the portion of human VEGF-D from residue 93 to201, i.e., with N- and C-terminal regions removed, was amplified bypolymerase chain reaction with Pfu DNA polymerase, using as template aplasmid comprising full-length human VEGF-D cDNA. The amplified DNAfragment, the sequence of which was confirmed by nucleotide sequencing,was then inserted into the expression vector pEFBOSSFLAG to give rise toa plasmid designated pEFBOSVEGFDΔNΔC. The pEFBOSSFLAG vector containsDNA encoding the signal sequence for protein secretion from theinterleukin-3 (IL-3) gene and the FLAG™ octapeptide. The FLAG™octapeptide can be recognized by commercially available antibodies suchas the M2 monoclonal antibody (IBI/Kodak). The VEGF-D PCR fragment wasinserted into the vector such that the IL-3 signal sequence wasimmediately upstream from the FLAG™ sequence, which was in turnimmediately upstream from the VEGF-D sequence. All three sequences werein the same reading frame, so that translation of mRNA resulting fromtransfection of pEFBOSVEGFDΔNΔC into mammalian cells would give rise toa protein which would have the IL-3 signal sequence at its N-terminus,followed by the FLAG™ octapeptide and the VEGF-D sequence. Cleavage ofthe signal sequence and subsequent secretion of the protein from thecell would give rise to a VEGF-D polypeptide which is tagged with theFLAG™ octapeptide adjacent to the N-terminus. This protein wasdesignated VEGFDΔNΔC.

In addition, a second plasmid was constructed, designatedpEFBOSVEGFDfullFLAG, in which the full-length coding sequence of humanVEGF-D was inserted into pEFBOSIFLAG such that the sequence for theFLAG™ octapeptide was immediately downstream from, and in the samereading frame as, the coding sequence of VEGF-D. The plasmid pEFBOSIFLAGlacks the IL-3 signal sequence, so secretion of the VEGF-D/FLAG fusionprotein was driven by the signal sequence of VEGF-D. pEFBOSVEGFDfullFLAGwas designed to drive expression in mammalian cells of full-lengthVEGF-D which was C-terminally tagged with the FLAG™ octapeptide. Thisprotein is designated VEGFDfullFLAG, and is useful for the study ofVEGF-D biosynthesis.

Analysis of the Post-Translational Processing of VEGF-D

To examine whether the VEGF-D polypeptide is processed to give a matureand fully active protein, pEFBOSVEGFDfullFLAG was transientlytransfected into COS cells (Aruffo and Seed, 1987). Expression in COScells followed by biosynthetic labeling with ³⁵S-methionine/cysteine andimmunoprecipitation with M2 gel has demonstrated species ofapproximately 43 kD (fA) and 25 kD(fB) (FIG. 12A). These bands areconsistent with the notion that VEGF-D is cleaved to give a C-terminalfragment (FLAG™ tagged) and an internal peptide (correspondingapproximately to the VEGFDΔNΔC protein). Reduction of theimmunoprecipitates (M2*) gives some reduction of the fA band, indicatingthe potential for disulphide linkage between the two fragments.

Expression and Purification of Internal VEGF-D Polypeptide

Plasmid pEFBOSVEGFDΔNΔC was used to transiently transfect COS cells bythe DEAE-Dextran method as described previously (Aruffo and Seed, 1987).The resulting conditioned cell culture medium (approximately 150 ml),collected after 7 days of incubation, was subjected to affinitychromatography using a resin to which the M2 monoclonal antibody hadbeen coupled. In brief, the medium was run batch-wise over a 1 ml M2antibody column for approximately 4 hours at 4° C. The column was thenwashed extensively with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl beforeelution with free FLAG™ peptide at 25 μg/ml in the same buffer. Theresulting material was used for the bioassays described below.

In order to detect the purified VEGFDΔNΔC, fractions eluted from the M2affinity column were subjected to Western blot analysis. Aliquots of thecolumn fractions were combined with 2×SDS-PAGE sample buffer, boiled,and loaded onto a 15% SDS polyacrylamide gel. The resolved fractionswere transferred to nitrocellulose membrane and non-specific bindingsites blocked by incubation in Tris/NaCl/Tween 20 (TST) and 10% skimmilk powder (BLOTTO). Membranes were then incubated with monoclonalantibody M2 or control antibody at 3 μg/ml for 2 h at room temperature,followed by extensive washing in TST. Membranes were then incubated witha secondary goat anti-mouse HRP-conjugated antiserum for 1 h at roomtemperature, followed by washing in TST buffer. Detection of the proteinspecies was achieved using a chemiluminescent reagent (ECL, Amersham)(FIG. 12B).

Under non-reducing conditions a species of molecular weightapproximately 23 kD (VEGFDΔNΔC) was detected by the M2 antibody. This isconsistent with the predicted molecular weight for this internalfragment (12,800) plus N-linked glycosylation; VEGFDΔNΔC contains twopotential N-linked glycosylation sites. A species of approximately 40 kDwas also detected, and may represent a non-covalent dimer of the 23 kDprotein (VEGFDΔNΔC).

Bioassays

The bioassay for the capacity of polypeptides to bind to VEGF receptor-2is described in detail in Example 7. Aliquots of fractions eluted fromthe M2 affinity column, containing the VEGFDΔNΔC protein, were dilutedin medium and tested in the VEGFR2 bioassay as previously described.Fraction #3 from the affinity column, which was shown to contain thepurified VEGFDΔNΔC protein (FIG. 12B), demonstrated a clear ability toinduce proliferation of the bioassay cell line to a dilution of 1/100 ofthe purified fraction (FIG. 13). In comparison, the void volume of theaffinity column (fraction #1) showed no activity, whereas the originalVEGFDΔNΔC conditioned medium gave only weak activity.

The vascular permeability assay (Miles and Miles, 1952) is described inbrief in Example 8. Aliquots of purified VEGFDΔNΔC, and samples of thevoid volume from the M2 affinity column (negative control) were combinedwith medium and injected intradermally into the skin of guinea pigs. Theregions of skin at the sites of injections were excised, andextravasated dye was eluted. The absorbance of the extravasated dye at620 nm arising from injection of purified VEGFDΔNΔC was 0.131±0.009. Incomparison, the value for absorbance arising from injection of a sampleof the void volume was 0.092±0.020. Therefore, VEGFDΔNΔC inducedvascular permeability, hut the effect was only marginal.

Due to its ability to bind to VEGFR2, and its lower induction ofvascular permeability compared to full length VEGF-D, VEGF-DΔNΔC may besaid to relatively decrease the induction of vascular permeability byVEGF-D through competitive inhibition. In this sense, the VEGF-DΔNΔCfragment may be thought of as an antagonist for VEGF-D as regards theinduction of vascular permeability.

SUMMARY

Two factors have led us to explore internal fragments of VEGF-D forenhanced activity. Firstly, it is the central region of VEGF-D whichexhibits amino acid homology with all other members of the VEGF family.Secondly, proteolytic processing which gives rise to internal bioactivepolypeptides occurs for other growth factors such as PDGF-BB. Inaddition, the activity seen with the full length VEGF-D protein in COScells was lower than for the corresponding conditioned medium fromVEGF-A transfected COS cells.

It was predicted that the mature VEGF-D sequence would be derived from afragment contained within residues 92-205, with cleavage at FAÂTFY andIIRR̂SIQI. Immunoprecipitation analysis of VEGF-DfullFLAG expressed inCOS cells produced species consistent with the internal proteolyticcleavage of the VEGF-D polypeptide at these sites. Therefore, atruncated form of VEGF-D, with the N- and C-terminal regions removed(VEGFDΔNΔC), was produced and expressed in COS cells. This protein wasidentified and purified using the M2 antibody. The VEGFDΔNΔC protein wasalso detected by the A2 antibody, which recognizes a peptide within the92-205 fragment of VEGF-D (not shown). VEGFDΔNΔC was evaluated by theVEGFR2 bioassay and the Miles vascular permeability assay, and shown tobind to and activate the VEGFR2 receptor in a bioassay designed todetect cross-linking of the VEGFR2 extracellular domain. Induction ofvascular permeability by this polypeptide in a Miles assay was at bestmarginal, in contrast to the effect of VEGF-A.

Example 10 VEGF-D Binds to and Activates VEGFR-3

The human VEGF-D cDNA was cloned into baculovirus shuttle vectors forthe production of recombinant VEGF-D. In addition to baculoviral shuttlevectors, which contained the unmodified VEGF-D cDNA (referred to as“full length VEGF-D”) two baculoviral shuttle vectors were assembled, inwhich the VEGF-D cDNA was modified in the following ways.

In one construct (referred to as “full length VEGF-D-H₆”) a C-terminalhistidine tag was added. In the other construct the putative N- andC-terminal propeptides were removed, the melittin signal peptide wasfused in-frame to the N-terminus, and a histidine tag was added to theC-terminus of the remaining VEGF homology domain (referred to as “ΔNΔc-MELsp-VEGF-D-H₆”).

For each of the three constructs, baculoviral clones of two or threeindependent transfections were amplified. The supernatant of High Five(HF) cells was harvested 48 h post infection with high titer virusstocks. The supernatant was adjusted to pH 7 with NaOH and diluted withone volume of D-MEM (0.2% FCS).

The samples were tested for their ability to stimulate tyrosinephosphorylation of VEGFR-3 (Flt-4 receptor) on NIH3T3 cells, asdescribed by Joukov et al., 1996. The supernatant of uninfected cellsand the supernatant of cells infected with the short splice variant ofVEGF-C, which does not stimulate tyrosine phosphorylation of VEGFR-3,were used as negative controls. VEGF-C modified in the same way asΔNΔC-melSP-VEGF-D-H, was used as positive control. The results are shownin FIG. 14.

The appearance of new bands at 125 and 195 kD indicates phosphorylation,and hence activation, of the receptor.

Example 11 VRGF-D Binds to and Activate VEGFR-2

Modified and unmodified human VEGF-D cDNA was cloned into baculovirusshuttle vectors for the production of recombinant VEGF-D as described inExample 10.

For each of the three constructs full length VEGF-D, full lengthVEGF-D-H₆, and ΔNΔC-melSP-VEGF-D-H₆, baculoviral clones of two or threeindependent transfections were amplified. The supernatant of High Five(HF) cells was harvested 48 hours post infection with high titer virusstocks. The supernatant was adjusted to pH 7 with NaOH and diluted withone volume of D-MEM (0.2% FCS).

The supernatants conditioned with the histidine-tagged proteins weretested for their ability to stimulate tyrosine phosphorylation of theKDR receptor according to Joukov et al., 1996. KDR is the human homologof flk1 (VEGFR-2).

The supernatant of uninfected cells and the supernatant of cellsinfected with the VEGF-C 156S mutant, which does not stimulate KDR, wereused as negative controls. VEGF₁₆₅ and VEGF-C modified in the same wayas ΔNΔC-melSP-VEGF-D-H₆ were used as positive controls. The results areshown in FIG. 15.

The appearance of a new band at approximately 210 kD indicatesphosphorylation, and hence activation, of the receptor.

Example 12 Analysis of VEFG-D Gene Expression

In order to characterize the pattern of VEGF-D gene expression in thehuman and in mouse embryos, VEGF-D cDNAs were used as hybridizationprobes for Northern blot analysis of polyadenylated human RNA and for insitu hybridization analysis with mouse embryos.

Gene Expression in the Adult Human

A 1.1 kb fragment of the human VEGF-D cDNA shown in FIG. 4 (SEQ ID NO:4)spanning from the EcoRV site to the 3′-terminus (nucleotides 911 to2029) was labeled with [α-³²P]dATP using the Megaprime DNA labelingsystem (Amersham) according to manufacturer's instructions. This probewas used to screen human multiple tissue northern blots (Clontech) byhybridization, also according to manufacturer's instructions. Theseblots contained polyadenylated RNA obtained from tissues of adult humanswho were apparently free of disease. Autoradiography with the labeledblots revealed that VEGF-D mRNA was most abundant in heart, lung, andskeletal muscle. VEGF-D mRNA was of intermediate abundance in spleen,ovary, small intestine, and colon, and was of low abundance in kidney,pancreas, thymus, prostate, and testis. No VEGF-D mRNA was detected inRNA from brain, placenta, liver, or peripheral blood leukocytes. In mostof the tissues where VEGF-D mRNA was detected the size of the transcriptwas 2.3 kb. The only exception was skeletal muscle, where two VEGF-Dtranscripts of 2.3 kb and 2.8 kb were detected. In skeletal muscle the2.3 kb transcript was more abundant than the 2.8 kb transcript.

Gene Expression in Mouse Embryos

In order to generate an antisense RNA probe for mouse VEGF-D mRNA, themouse VEGF-D2 cDNA shown in FIG. 7 (SEQ ID NO:7) was inserted into thetranscription vector pBluescriptIIKS+ (Stratagene). The resultingplasmid was digested to completion with the restriction endonucleaseFokI and then used as template for an in vitro transcription reactionwith T3 RNA polymerase. This transcription reaction gave rise to anantisense RNA probe for VEGF-D mRNA which was complementary in sequenceto the region of the VEGF-D2 cDNA (FIG. 7) from the 3′-terminus to theFokI cleavage site closest to the 3′-terminus (nucleotides 1135 to 700).This antisense RNA probe was hybridized at high stringency withparaffin-embedded tissue sections generated from mouse embryos atpost-coital day 15.5. Hybridization and washing were essentially asdescribed previously (Achen et al., 1995).

After washing and drying, slides were exposed to autoradiography filmfor six days.

Development of the autoradiography film revealed that VEGF-D mRNA islocalized in the developing lung of post-coital day 15.5 embryos. Thesignal for VEGF-D mRNA in the lung was strong and highly specific.Control hybridizations with sense probe gave no detectable background inlung or any other tissue.

SUMMARY

The VEGF-D gene is broadly expressed in the adult human, but iscertainly not ubiquitously expressed. Strongest expression was detectedin heart, lung and skeletal muscle. In mouse embryos at post-coital day15.5, strong and specific expression of the VEGF-D gene was detected inthe lung. These data suggest that VEGF-D may play a role in lungdevelopment, and that expression of the VEGF-D gene in lung persists inthe adult, at least in humans. Expression of the gene in other tissuesin the adult human suggests that VEGF-D may fulfill other functions inother adult tissues.

Example 13 VEGF-D in Mitogenic for Endothelial Cells

Some members of the VEGF family of proteins, namely VEGF-A (Leung etal., 1989) and VEGF-B (Olofsson et al., 1996), are mitogenic forendothelial cells. In order to test the mitogenic capacity of VEGFDΔNΔCfor endothelial cells, this protein was expressed and purified byaffinity chromatography as described in Example 9. Fraction #3, elutedfrom the M2 affinity column, which contained VEGFDΔNΔC, was diluted 1 in10 in cell culture medium containing 5% serum and applied to bovineaortic endothelial cells (BAEs) which had been propagated in mediumcontaining 10% serum. The BAEs had been seeded in 24-well dishes at adensity of 10,000 cells per well the day before addition of VEGFDΔNΔC,and 3 days after addition of this polypeptide the cells were dissociatedwith trypsin and counted. Purified VEGF-A was included in the experimentas positive control. Results are shown in FIG. 16. The addition offraction #3 to the cell culture medium led to a 2.4-fold increase in thenumber of BAEs after 3 days of incubation, a result which was comparableto that obtained with VEGF-A. Clearly VEGFDΔNΔC is mitogenic forendothelial cells.

Example 14 Localization of the VEGF-D Gene on Human Chromosomes

In order to generate hybridization probes for localization of the VEGF-Dgene on human chromosomes, a human genomic DNA clone for VEGF-D wasisolated from a human genomic DNA library (Clontech). The genomiclibrary was screened by hybridization with the human VEGF-D cDNA shownin FIG. 4, using standard methods (Sambrook et al., 1989). One of theclones thus isolated was shown to contain part of the VEGF-D gene byhybridization to numerous oligonucleotides which were derived insequence from the human VEGF-D cDNA. A region of the genomic clone,approximately 13 kb in size, was purified from agarose gel, labeled bynick-translation with biotin-14-dATP and hybridized in situ at a finalconcentration of 20 ng/μl to metaphases from two normal human males. Thefluorescence in situ hybridization (FISH) method was modified from thatpreviously described (Callen et al., 1990) in that chromosomes werestained before analysis with propidium iodide (as counterstain) and DAPI(for chromosome identification). Images of metaphase preparations werecaptured by a cooled CCD camera, using the CytoVision Ultra imagecollection and enhancement system (Applied Imaging Int. Ltd.). FISHsignals and the DAPI banding pattern were merged for analysis.

Fifteen metaphases from the first normal male were examined forfluorescent signal. Ten of the metaphases showed signal on one chromatid(3 cells) or both chromatids (7 cells) of the X chromosome in bandp22.1. There was a total of 9 non-specific background dots observed inthese 15 metaphases. A similar result was obtained from hybridization ofthe probe to 15 metaphases from the second normal male, where signal wasobserved at Xp22.1 on one chromatid in 7 cells and on both chromatids in4 cells. In conclusion, the human VEGF-D gene is located on the Xchromosome in band p22.1.

Example 15 Localization of the Murine VEGF-D Gene on Mouse Chromosomes

The mouse chromosomal location of the VEGF-D gene was determined byinterspecific backcross analysis using progeny generated by mating(C57BL/6J×Mus spretus) F1 females and CB7BL/67 males as describedpreviously (Copeland and Jenkins, 1991). This interspecific backcrossmapping panel has been typed for over 2400 loci that are welldistributed among all the autosomes as well as the X chromosome(Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digestedwith several enzymes and analyzed by Southern blot hybridization forinformative restriction fragment length polymorphisms (RFLPs) using a1.3 kb mouse VEGF-D cDNA probe essentially as described (Jenkins et al.1982). Fragments of 7.1, 6.3, 4.7, 2.5 and 2.2 kb were detected inTaqI-digested C57BL/6J DNA and major fragments of 7.1, 3.7, 2.7 and 2.2kb were detected in TaqI-digested M. spretus DNA. The presence orabsence of the 3.7 and 2.7 TaqI M. spretus-specific fragments, whichcosegregated, was followed in backcross mice. The mapping resultsindicated that the VEGF-D gene is located in the distal region of themouse X chromosome linked to Bik, DxPasI and Ptmb4. Although 89 micewere analyzed for every marker, up to 133 mice were typed for some pairsof markers. Each locus was analyzed in pairwise combinations forrecombination frequencies using the additional data. The ratios of thetotal number of mice exhibiting recombinant chromosomes to the totalnumber of mice analyzed for each pair of loci and the most likely geneorder are: centromere-Btk-14/121-DxPasI-3/99-VEGF-D-5/133-Ptmb4. Therecombination frequencies [expressed as genetic distances incentiMorgans (cM) ±the standard error], calculated using Map Manager(version 2.6.5), are-Btk-11.6+/−2.9-DxPasI-3.0+/−1.7-VEGF-D-3.8+/−1.7-Ptmb4. A descriptionof the probes and RFLPs for the loci linked to the VEGF-D gene,including Btk, DxPasI and Ptmb4, has been reported previously (Hacfligeret al., 1992; Holloway et al., 1997).

We have compared our interspecific map of the X chromosome with acomposite mouse linkage map that reports the map location of manyuncloned mutations (provided from Mouse Genome Database, a computerizeddatabase maintained at The Jackson Library, Bar Harbor, Me.). The VEGF-Dgene mapped in a region of the composite map that lacks mouse mutationswith a phenotype that might be expected for an alteration in the locusfor an endothelial cell mitogen. The distal region of the mouseX-chromosome shares a region of homology with the short arm of the humanX chromosomes (Mouse Genome Database). The placement of the VEGF-D genein this interval in mouse suggests that the human homolog will map toXp22. This is consistent with our FISH analysis which has localized thehuman gene to Xp22.1.

Numerous disease states are caused by mutations in unknown genes whichhave been mapped to Xp22.1 and the positions immediately surroundingthis region in the human. These disease states include Kallmannsyndrome, ocular albinism (Nettleship-Falls type), ocular albinism andsensorineural deafness, Partington syndrome, spondyloepiphysealdysplasia (late), retinitis pigmentosa 15, gonadal dysgenesis (XY femaletype), Nance-Horan cataract-dental syndrome, retinoschisis,Charcot-Marie-Tooth disease, F-cell production, hypomagnesemia,keratosis follicularis spinulosa decalvans, Coffin-Lowry syndrome,corneal dermoids, hypophosphatemia, agammaglobulinemia, Aicardisymdrome, hereditary hypophosphatemia II, mental retardation(non-dysmorphic), Opitz G syndrome, pigment disorder (reticulate),dosage-sensitive sex reversal, adrenal hypoplasia, retinitispigmentosa-6, deafness 4 (congenital sensorineural) and Wilson-Turnersyndrome. The positions of the genes involved in these disease statesare documented in the OMIM gene map which is edited by Dr. VictorMcKusick and colleagues at Johns Hopkins University (USA).

Bioassays to Determine the Function of VEGF-D

Other assays are conducted to evaluate whether VEGF-D has similaractivities to VEGF in relation to endothelial cell function,angiogenesis and wound healing. Further assays may also be performed,depending on the results of receptor binding distribution studies.

I. Assays of Endothelial Cell Function a) Endothelial Cell Proliferation

Endothelial cell growth assays are performed by methods well known inthe art, e.g., those of Ferrara & Henzel (1989), Gospodarowicz et al.(1989), and/or Claffey et al., Biochim. Biophys. Acta, 1995 1246 1-9.

b) Cell Adhesion Assay

The effect of VEGF-D on adhesion of polmorphonuclear granulocytes toendothelial cells is tested.

c) Chemotaxis

The standard Boyden chamber chemotaxis assay is used to test the effectof VEGF-D on chemotaxis.

d) Plasminogen Activator Assay

Endothelial cells are tested for the effect of VEGF-D on plasminogenactivator and plasminogen activator inhibitor production, using themethod of Pepper et al. (1991).

e) Endothelial Cell Migration Assay

The ability of VEGF-D to stimulate endothelial cells to migrate and formtubes is assayed as described in Montesano et al. (1986). Alternatively,the three-dimensional collagen gel assay described by Joukov et al.(1996) or a gelatinized membrane in a modified Boyden chamber (Glaser etal., 1980) may be used.

II Angiogenesis Assay

The ability of VEGF-D to induce an angiogenic response in chickchorioallantoic membrane is tested as described in Leung et al. (1989).Alternatively the rat cornea assay of Rastinejad et al. (1989) may beused; this is an accepted method for assay of in vivo angiogenesis, andthe results are readily transferrable to other in vivo systems.

III Wound Healing

The ability of VEGF-D to stimulate wound healing is tested in the mostclinically relevant model available, as described in Schilling et al.(1959) and utilized by Hunt et al. (1967).

IV The Haemopoietic System

A variety of in vitro and in vivo assays using specific cell populationsof the haemopoietic system are known in the art, and are outlined below.In particular a variety of in vitro murine stem cell assays usingfluorescence-activated cell sorter purified cells are particularlyconvenient:

a) Repopulating Stem Cells

These are cells capable of repopulating the bone marrow of lethallyirradiated mice, and have the Lin⁻, Rh^(h1), Ly-6A/E, c-kit⁺ phenotype.VEGF-D is tested on these cells either alone, or by co-incubation withother factors, followed by measurement of cellular proliferation by³H-thymidine incorporation.

b) Late Stage Stem Cells

These are cells that have comparatively little bone marrow repopulatingability, but can generate D13 CFU-S. These cells have the Lin⁻, Rh^(h1),Ly-6A/E⁺, c-kit⁺ phenotype. VEGF-D is incubated with these cells for aperiod of time, injected into lethally irradiated recipients, and thenumber of D13 spleen colonies enumerated.

c) Progenitor-Enriched Cells

These are cells that respond in vitro to single growth factors and havethe Lin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺ phenotype. This assay will show ifVEGF-D can act directly on haemopoietic progenitor cells. VEGF-D isincubated with these cells in agar cultures, and the number of coloniespresent after 7-14 days is counted.

V Atherosclerosis

Smooth muscle cells play a crucial role in the development or initiationof atherosclerosis, requiring a change of their phenotype from acontractile to a synthetic state. Macrophages, endothelial cells, Tlymphocytes and platelets all play a role in the development ofatherosclerotic plaques by influencing the growth and phenotypicmodulations of smooth muscle cell. An in vitro assay using a modifiedRose chamber in which different cell types are seeded on to oppositecoverslips measures the proliferative rate and phenotypic modulations ofsmooth muscle cells in a multicellular environment, and is used toassess the effect of VEGF-D on smooth muscle cells.

VI Metastasis

The ability of VEGF-D to inhibit metastasis is assayed using the Lewislung carcinoma model, for example using the method of Cao et al. (1995).

VII VEGF-D in Other Cell Types

The effects of VEGF-D on proliferation, differentiation and function ofother cell types, such as liver cells, cardiac muscle and other cells,endocrine cells and osteoblasts can readily be assayed by methods knownin the art, such as ³H-thymidine uptake by in vitro cultures. Expressionof VEGF-D in these and other tissues can be measured by techniques suchas Northern blotting and hybridization or by in situ hybridization.

VIII Construction of VEGF-D Variants and Analogs

VEGF-D is a member of the PDGF family of growth factors which exhibits ahigh degree of homology to the other members of the PDGF family. VEGF-Dcontains eight conserved cysteine residues which are characteristic ofthis family of growth factors. These conserved cysteine residues formintra-chain disulfide bonds which produce the cysteine knot structure,and inter-chain disulfide bonds that form the protein dimers which arecharacteristic of members of the PDGF family of growth factors. VEGF-Dwill interact with protein tyrosine kinase growth factor receptors.

In contrast to proteins where little or nothing is known about theprotein structure and active sites needed for receptor binding andconsequent activity, the design of active mutants of VEGF-D is greatlyfacilitated by the fact that a great deal is known about the activesites and important amino acids of the members of the PDGF family ofgrowth factors.

Published articles elucidating the structure/activity relationships ofmembers of the PDGF family of growth factors include for PDGF: Oestmanet al., J. Biol. Chem., 1991 26 10073-10077; Andersson et al., J. Biol.Chem., 1992 26 11260-1266; Oefner et al., EMBO J., 1992 11 3921-3926;Flemming et al., Molecular and Cell Biol., 1993 13 4066-4076 andAndersson et al., Growth Factors, 1995 12 159-164; and for VEGF: Kim etal., Growth Factors, 1992 7 53-64; Pötgens et al., J. Biol. Chem., 1994269 32879-32885 and Claffey et al., Biochem. Biophys. Acta, 1995 12461-9. From these publications it is apparent that because of the eightconserved cysteine residues, the members of the PDGF family of growthfactors exhibit a characteristic knotted folding structure anddimerization, which result in formation of three exposed loop regions ateach end of the dimerized molecule, at which the active receptor bindingsites can be expected to be located.

Based on this information, a person skilled in the biotechnology artscan design VEGF-D mutants with a very high probability of retainingVEGF-D activity by conserving the eight cysteine residues responsiblefor the knotted folding arrangement and for dimerization, and also byconserving, or making only conservative amino acid substitutions in thelikely receptor sequences in the loop 1, loop 2 and loop 3 region of theprotein structure.

The formation of desired mutations at specifically targeted sites in aprotein structure is considered to be a standard technique in thearsenal of the protein chemist (Kunkel et al., Methods in Enzymol., 1987154 367-382). Examples of such site-directed mutagenesis with VEGF canbe found in Pötgens et al., J. Biol. Chem., 1994 269 32879-32885 andClaffey et al., Biochim. Biophys. Acta, 1995 1246 1-9. Indeed,site-directed mutagenesis is so common that kits are commerciallyavailable to facilitate such procedures (eg. Promega 1994-1995 Catalog.,Pages 142-145).

The endothelial cell proliferating activity of VEGF-D mutants can bereadily confirmed by well established screening procedures. For example,a procedure analogous to the endothelial cell mitotic assay described byClaffey et al., (Biochim. Biophys. Acta., 1995 1246 1-9) can be used.Similarly the effects of VEGF-D on proliferation of other cell types, oncellular differentiation and on human metastasis can be tested usingmethods which are well known in the art.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by reference.

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1-76. (canceled)
 77. A pharmaceutical composition comprising apolypeptide that has at least one activity selected from the groupconsisting of stimulation of endothelial cell proliferation, stimulationof vascular permeability, binding to extracellular domain of VEGFR-2,and binding to extracellular domain of VEGFR-3, wherein the polypeptidecomprises a fragment of the amino acid sequence of SEQ ID NO: 5, whereinsaid fragment and said polypeptide lack amino acids of SEQ ID NO: 5 thatare N- and C-terminal to residues 92-205 of SEQ ID NO:
 5. 78. Thepharmaceutical composition of claim 77, wherein the polypeptidecomprises at least amino acids 101-196 of SEQ ID NO:
 5. 79. Thepharmaceutical composition of claim 77, wherein the polypeptide lacksamino acids of SEQ ID NO: 5 that are N- and C-terminal to residues93-201 of SEQ ID NO:
 5. 80. The pharmaceutical composition of claim 77,wherein the polypeptide is present in the composition as a homodimer.81. The pharmaceutical composition of claim 79, further comprising aFLAG peptide linked to the polypeptide.